production of citric acid , acetic acid and gluconic acid... CITRIC ACID. Citric acid is a weak organic acid found in citrus fruits. It is naturally found in fruits such as lemon, orange, pineapple, plum, and pear. - Molecular formula is C6H8O7 and belongs to the carboxylic acids groups. - Stronger acid compared to other typical carboxylic acid. Produced by fermentation and suitable pH is around 3-6.  Citric acid is ( 2- hydroxy-1,2,3 propane tricarboxylic acid).  Citric acid is excreted from the cells in response to unfavorable intracellular condition caused by increased levels of tricarboxylic acids (TCA) A crucial prerequisite for overflow of citric acid from A. niger cells is therefore increased level of Krebs cycle intermediates caused by anaplerotic reactions. ACETIC ACID • Acetic Acid is systematically named as ethanoic acid. • It is a colorless liquid organic compound. • It has a pungent/ vinegar-like odor. • Glacial acetic acid is the pure form of acetic acid (99.98%). • Vinegar is product of Acetic acid. The first vinegar was spoiled wine. • It has melting point 16 to 17°C; 61 to 62°F. GLUCONIC ACID. Introduction: Gluconic acid is an organic compound with molecular formula C6H12O7 and condensed structural formula HOCH2 (CHOH)4COOH. It is one of the 16 stereoisomers of 2,3,4,5,6-pentahydroxyhexanoic acid. In aqueous solution at delicately acidic pH, gluconic acid forms the gluconate ion. Gluconic Acid is the carboxylic acid formed by the oxidation of the first carbon of glucose with antiseptic and chelating properties. Gluconic acid, found abundantly in plant, honey and wine, can be prepared by fungal fermentation process commercially. This agent and its derivatives can used in formulation of pharmaceuticals, cosmetics and food products as additive or buffer salts. Aqueous gluconic acid solution contains cyclic ester glucono delta lactone structure, which chelates metal ions and forms very stable complexes. In alkaline solution, this agent exhibits strong chelating activities towards anions, i.e. calcium, iron, aluminum, copper, and other heavy metals.

1. BIOPROCESS ENGINEERING
Production of Citric , Acetic and Gluconic Acid.
AYUSHI SOMVANSHI
BT190203

2.  INTRODUCTION:
- Citric acid is a weak organic acid found in citrus fruits. It is naturally found in fruits
such as lemon, orange, pineapple, plum, and pear.
- Molecular formula is C6H8O7 and belongs to the carboxylic acids groups.
- Stronger acid compared to other typical carboxylic acid.
- Produced by fermentation and suitable pH is around 3-6. Citric acid is ( 2- hydroxy-
1,2,3 propane tricarboxylic acid).
- Citric acid is excreted from the cells in response to
unfavorable intracellular condition caused by increased
levels of tricarboxylic acids (TCA)
- A crucial prerequisite for overflow of citric acid from A. niger
cells is therefore increased level of Krebs cycle intermediates
caused by anaplerotic reactions Molecular weight: 192g

3. Beet Molasses
• The source of sugar for
microbial production of
citric acid.
• Low cost and high sugar
content.
• Low content of trace
metals.
• Acts as carbon source of
the fermentation.
Microorganis
m
• Mycomycetes of A.niger
species can produce high
yield.
• Consequence of
incomplete respiration.
Aspergillus
niger
• Filamentous ascomycetes
fungus.
• Maintained at pH 4.5 and
temperature at 45℃
• The best strain for citric
acid production.
• Scientific classification of
A.niger

4.  APPLICATION IN INDUSTRY:
– Beverages
– Food
– Pharmaceutical
– Agriculture
– Metal Industry.
 PRODUCTION:
 Fermentation is the most economical and widely used for citric acid production.
 The industrial citric acid production can be carried in three different ways:
Surface Fermentation
Submerged Fermentation
Solid State Fermentation

5. SURFACE FERMENTATION:
Surface fermentation using Aspergillus niger may be done on rice bran as
is the case in Japan, or in liquid solution in flat aluminum or stainless steel
pans.
Special strains of Aspergillus niger which can produce citric acid despite
the high content of trace metals in rice bran are used.
Molasses
substrate(15-
20 % of
sucrose, added
nutrients)
acidified with,
phosphoric
acid to a pH
6.0 - 6.5 and
heated at
temperature
110 ºC for 15
to 45 min.
Potassium
hexacyanoferrat
e is added to the
hot substrate, to
precipitate or
complex trace
metals [Fe, Mn,
Zn] and to act in
excess as a
metabolic
inhibitor
restricting
growth and
promoting acid
production.
Inoculation is
performed in
two ways, as a
suspension of
conidia added to
the cooled
medium, or as a
dry conidia
mixed with
sterile air and
spread as an
aerosol over the
trays .
The
temperatur
e is kept
constant at
30 ºC
during the
fermentati
on by
means of
air current.
Within 24
hours after
inoculation, the
germinating
spores start
forming a 2-3
cm cover
blanket of
mycelium
floating on the
surface of the
substrate.
As a result of
the uptake of
ammonium
ions the pH of
the substrate
falls to 2.0
.The fully
developed
mycelium
floats as a
thick white
layer on the
nutrient
solution.
The
fermentati
on process
stops after
8 - 14
days.
Recovery of
mycelium
to extract
citric acid.

6.  SOLID STATE FERMENTATION:
It is simplest method for citric acid production.
Solid state fermentation is also known as koji process, was first developed in
Japan.
Citric acid production reached a maximum(88g/kg dry matter)when
fermentation as carried out with cassava having initial moisture of 62% at 26℃for
120 hours.
The solid
substrate is
soaked with
water up to 65 -
70 % of water
content. After the
removal of excess
water, the mass
undergoes a
steaming process
Sterile starch
paste is
inoculated by
spreading
Aspergillus niger
conidia in the
form of aerosol
or as a liquid
conidia
suspension on
the substrate
surface
The pH of the
substrate is about
5 to 5.5, and
incubation
temperature 28
to 30 ºC.
Growth can be
accelerated by
adding α-amylase,
although the
fungus can
hydrolyze starch
with its own α-
amylase. During
the citric acid
production pH
dropped to values
below 2
The solid state
surface process
takes 5 to 8 days at
the end of which
the entire is
extracted with hot
water. On other
cases, mechanical
passes are also used
to obtain more
citric acid from the
cells

7.  SUBMERGED FERMENTATION:
In this case , the strains are inoculated of about 15cm depth in fermentation
tank.
The culture is enhanced by giving aeration using air bubbles
And its allowed to grow for about 5 to 14 days at 27℃ to 33℃
The citric acid produced in the fermentation tank and it is purified.
Beet molasses
substrate (12 - 15
%. reducing sugar
content ), Nutritive
salts, such as
ammonium nitrate
or potassium
dihydrogen
phosphate are
added. pH of
substrate is
maintained at 5.5
to 5.9.
The process can
usually run in one
or two stages,
using hydrophilic
spores suspensions
or germinated
conidia from the
propagator stage .
Amounts of spores
are 5 to 25 x 106
per litre of
substrate.
The
development
of the hyphae
and the
aggregation
generally
requires a
period from 9
to 25 hours at
temperature of
32ºC.
Mycelia
aggregation
and spherical
pellets, the
productive
form can be
detected after
24 hrs. of
inoculation.
The change of
pH in this
phase is from
5.5 to 3.5, for
beet molasses
substrate, and
to 2.2 for the
sucrose
substrate.
Fermentatio
n last upto
6-8 days
and later
citric acid is
purified
from
mycelium.

10. Uses Of Citric Acid:
 It is used in the production of carbonated beverages.
 As a chelating and sequestering agent in the tanning and textile industry.
 Citrate esters are used as plasticizer.
 It is abundantly used in food industry as an acidulent in the preparation of food items like
jams, preserved fruits and fruit juices etc.
 It is used in frozen foods to prevent its change in color and flavor.
 Metal painting industry.
 In the manufacture of astringent, hair rinsers and hair setting fluids.
 In beverage industry as a preservative to prevent oxidation of alcohol, emulsifier of dairy
products like cheese and ice creams.
 It is used as preservative and to prevent change in color, flavor and in the oxidation of
alcohol.

11. INTRODUCTION:
• Acetic Acid is systematically named as ethanoic acid.
• It is a colorless liquid organic compound.
• It has a pungent/ vinegar-like odor.
• Glacial acetic acid is the pure form of acetic acid (99.98%).
• Vinegar is product of Acetic acid. The first vinegar was spoiled wine.
• It has melting point 16 to 17°C; 61 to 62°F.
ACETIC ACID BACTERIA :
• Acetic acid bacteria is a gram-negative bacteria which oxidize sugar
or ethanol and produce acetic acid during fermentation.

12. • Acetic acid bacteria are rod-shaped & obligate aerobes.
• Pasteur in 1864 claimed that the transformation of wine into vinegar was
due to the veil of Mycoderma aceti on it surface pungency of acetic acid.
PROPERTIES OF ACETIC ACID:
It is a colorless liquid with pungent smell.
melting point 16-17 degree centigrade.
Boiling point 118.0 degree centigrade.
Glacial acetic acid is highly corrosive to metals.
Soluble in alcohol.
Miscible with water, glycerol ,ether , acetone and benzene.
Insoluble in carbon disulfide.

13. RAW MATERIALS:
o Biomass Feedstock (beach wood, Silicon tube, sieving, wood chips, green
crops, manure, farm and mill waste)
o Ethanol is used as substrate Microbes Acetic Acid Bacteria (Acetobacter
aceti ) used as bio reactor
o Cooked Grapes
MICROBES EMPLOYED:
o Genus Acetobacterium
commonly used- Acetobacterium aceti and Acetobactor xylinum
o Acetic Acid Bacteria (fruit flies and vinegar eels propagate AAB in
nature) o Genus Clostridium
commonly used- Clostridium thermoaceticum -anerobic thermophile

14. Trickling Generation Submerged liquid
fermentation

16. Place a wooden container with beach wood
sieving
Place a sprayer above the container
Supply the air in the container
Add a substrate in the container
Oxidation process
Vinegar will produced
Process with again continuous with TBV
80 TO 88% Vinegar will be produced
PH range- 2.5 to
3.2 temp 20⁰C to
30⁰C

19. Pour the substrate in Silicon Tube
Add a inoculum in the tube
Agitation is given
Oxidization process
After 72 hours
90% vinegar is produced
Temp 40⁰C to 42⁰C

21. Uses Of Acetic
Acid:
As a Coagulant for latex in rubber industry.
In the manufacture of Plastic (polyvinyl acetate) rayon
(cellulose acetate) and silk.
In medicine as a local irritant.
As a solvent in the laboratory for carrying out reactions.
In the manufacture of many organic compound like
acetone, acetates &ester.
Heat transfer liquids , Neutralizer , Fungicide and De-icers.
Textile finishes, vinyl plastics, latex paints and adhesives.

22. Introduction:
 Gluconic acid is an organic compound with molecular formula C6H12O7
and condensed structural formula HOCH2 (CHOH)4COOH.
 It is one of the 16 stereoisomers of 2,3,4,5,6-pentahydroxyhexanoic acid. In
aqueous solution at delicately acidic pH, gluconic acid forms the gluconate
ion.
 Gluconic Acid is the carboxylic acid formed by the oxidation of the
first carbon of glucose with antiseptic and chelating properties.
 Gluconic acid, found abundantly in plant, honey and wine, can be prepared
by fungal fermentation process commercially. This agent and its derivatives
can used in formulation of pharmaceuticals, cosmetics and food products as
additive or buffer salts.
 Aqueous gluconic acid solution contains cyclic ester glucono delta
lactone structure, which chelates metal ions and forms very stable complexes.
In alkaline solution, this agent exhibits strong chelating activities towards
anions, i.e. calcium, iron, aluminum, copper, and other heavy metals.

23.  Gluconic acid is produced from glucose. This, reaction is catalyzed by the enzyme
glucose oxidase.
 The gluconolactone formed at the end of the first step undergoes hydrolysis either
spontaneously or enzymatically to produce gluconic acid.
 During the first reaction, the hydrogen from FADH2 is transferred to oxygen leading to
the formation of H2O2 which is immediately split into water and oxygen by the
enzyme catalase, to prevent its antimicrobial activity.

24. Fermentation Process of Gluconic Acid:
 Gluconic acid is produced industrially by employing the fungus or the bacterium. In the former process
Aspergillus niger and in the later process Acetobacter suboxidans are used.
 A. niger employs glucose oxidase involving agent like FAD and lactonase in the presence of O2 resulting
in the formation of gluconic acid.
 On the other hand, Gluconobacter employs glucose dehydrogenase with coenzyme pyrroloquinoline
quinone (PQQ) and lactonase which help in dissipating hydrogen peroxide.

25. (a) Fungal Fermentation:
 Submerged fermentation process is employed for fungal fermentation.
Aspergillus niger is used as a microorganism. Either sporulated culture or
spores germinated in seed tank is used as inoculum. Each method has its own
advantages.
 For example – use of spore inoculum directly avoids the cost of installation
and operation of the seed tank, while use of germinated spores reduces
operating cycles for the main fermenter.
 Glucose is used as a solution or crystalline glucose or in the form of a syrup
prepared from starch or crude starchy material which are treated with
amylase and amyloglucosidase
 It is necessary to maintain maximum concentration of dissolved oxygen in
the form of solution by vigorous agitation with the help of turbido mixture or
cavitator.
 In the manufacture of calcium or sodium gluconate optimum temperature is
maintained at 28-30°C and pH 6.5 along with vigorous agitation and
aeration.

26. Recovery and Harvest:
 After fermentation, the fungal mycelium is separated by filtration, the separated
mycelium is used for the recovery of glucose. On the other hand, the filtrate is used for
the recovery of calcium or sodium gluconate.

27. (iv) Production of Pure Gluconic Acid:
 It is also possible to obtain gluconic acid in pure form by the following procedure,
where calcium gluconate is employed:
 Calcium is precipitated by the addition of sulfuric acid. Calcium sulfate thus
formed is separated by filtration.
 The filtrate is decolorized with activated charcoal.
 The acid solution is concentrated to 50 percent acid strength. The product thus
formed is a mixture of gluconic acid and lactones.
 The concentrate is then treated with a temperature ranging from 0°C-30°C. The
crystals that separate at this temperature consist of pure gluconic acid.
 When the concentrate is treated with a temperature ranging from 30°-70°C
crystals of lactones separate.
 Y-lactone crystals get separated when the concentrate is treated with 70°C and
above temperature.

28. (b) Bacterial Fermentation:
 Submerged fermentation process is used. The bacterium employed is Acetobacter
suboxydans or Gluconobacter suboxydans. Pure culture of the bacterium is raised
by repeated sub-culturing process which is used in the fermentative production
of gluconic acid and is produced either in the form of calcium gluconate or
sodium gluconate.
 Glucose is used as a substratum in the fermentation. If calcium gluconate is to be
produced 13-15% glucose can be added as a substrate because of low solubility
of calcium gluconate (4 g liter-1 at 30°C).
 Higher calcium gluconate levels are formed if glucose is used above 13% levels
which would spontaneously crystallize as calcium gluconate, and makes
purification difficult. If sodium gluconate is to be produced, a glucose
concentration of 28-30% can be used.
 The fermentation is carried out at a temperature of 28-30°C, pH 4.5-6.5 with a
high aeration rate of 1-1.5 vvm. The gluconic acid yield can be substantially
increased (90-95%) by increasing the solubility of oxygen which is achieved by
raising the pressure in the system.

29. Uses of Gluconic Acid:
Gluconic acid is used in the manufacture of metal, leather
and food.
Sodium gluconate is used as a sequestering agent in many
detergents.
Calcium gluconate is used in medicine.
Gluconolactone is used as baking powder and as an
additive.

30.  https://www.biotechnologynotes.com/industrial-biotechnology/citric-acid/citric-acid-structure-fermentation-
process-and-uses-in-food-industries-biotechnology/13744
 https://www.biotechnologynotes.com/industrial-biotechnology/citric-acid/acetic-acid-structure-fermentation-
process-and-uses-in-food-industries-biotechnology/13744
 https://www.biotechnologynotes.com/industrial-biotechnology/citric-acid/gluconic-acid-structure-
fermentation-process-and-uses-in-food-industries-biotechnology/13744
 Yuguo, Z., Zhao, W., & Xiaolong, C. (1999). Citric acid production from the mash of dried sweet
potato with its dregs by Aspergillus niger in an external-loop airlift bioreactor.
 Process Biochemistry, 35(3-4), 237-242. Berovic, M., & Legisa, M. (2007). Citric acid production.
Biotechnology annual review, 13, 303-343. Swain, M. R., Ray, R. C., & Patra, J. K. (2011).
 Citric acid: microbial production and applications in food and pharmaceutical industries. Citric Acid:
Synthesis, Properties and Applications, Edition, 1, 97-118. Gupta, S., & Sharma, C. B. (2002).
 Biochemical studies of citric acid production and accumulation by Aspergillus niger mutants. World
Journal of Microbiology and Biotechnology, 18(5), 379-383.